Federal Register, Volume 64 Issue 55 (Tuesday, March 23, 1999)

[Federal Register Volume 64, Number 55 (Tuesday, March 23, 1999)]
[Notices]
[Pages 14086-14096]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 99-7022]

[[Page 14085]]

_______________________________________________________________________

Part IV

Department of Health and Human Services

_______________________________________________________________________

Centers for Disease and Prevention

_______________________________________________________________________

Annual Submission of the Quantity of Nicotine Contained in Smokeless
Tobacco Products Manufactured, Imported, or Packaged in the United
States Requirement; Notice

Federal Register / Vol. 64, No. 55 / Tuesday, March 23, 1999 /
Notices

[[Page 14086]]

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Centers for Disease Control and Prevention

Notice Regarding Requirement for Annual Submission of the
Quantity of Nicotine Contained in Smokeless Tobacco Products
Manufactured, Imported, or Packaged in the United States

AGENCY: Centers for Disease Control and Prevention (CDC), Department of
Health and Human Services.

ACTION: Notice.

-----------------------------------------------------------------------

SUMMARY: This notice establishes a uniform protocol for the analysis of
nicotine, total moisture, and pH in smokeless tobacco products. This
protocol was designed to implement the requirement of the Comprehensive
Smokeless Tobacco Health Education Act (CSTHEA) of 1986 (15 U.S.C. 4401
et seq., Pub. L. 99-252), which requires that each entity
manufacturing, packaging, or importing smokeless tobacco products shall
annually provide the Secretary of Health and Human Services (HHS) with
a specification of the quantity of nicotine contained in each smokeless
tobacco product.

DATES: The first report of information is due June 30, 1999, with
subsequent submissions due by March 31 of each year.

ADDRESSES: The information shall be submitted to: Michael P. Eriksen,
Sc.D., Director, Office on Smoking and Health, National Center for
Chronic Disease Prevention and Health Promotion, Centers for Disease
Control and Prevention (CDC), 4770 Buford Highway, NE., Atlanta, GA
30341-3724.

FOR FURTHER INFORMATION CONTACT: Michael P. Eriksen, Sc.D., Director,
Office on Smoking and Health, telephone: (770) 488-5701.

SUPPLEMENTARY INFORMATION: According to a 1986 report of an Advisory
Committee to the Surgeon General, smokeless tobacco represents a
significant health risk, is not a safe substitute for cigarette
smoking, can cause cancer and a number of noncancerous oral conditions,
and can lead to nicotine addiction.
The Centers for Disease Control and Prevention's (CDC) Office on
Smoking and Health (OSH) has primary responsibility for the Department
of Health and Human Services' (HHS) tobacco and health program. The
overall goal of this program is to reduce death and disability
resulting from cigarette smoking and other forms of tobacco use through
programs of information, education, and research.
HHS uses the information collected to exercise its authority under
CSTHEA to conduct research on the addictive nature of nicotine and
general health effects of using smokeless tobacco. Nicotine data will
provide a more complete picture of the addictive nature of smokeless
tobacco products. Also, as authorized in the statute, HHS may report to
the Congress information regarding its current and proposed research
relative to nicotine levels in smokeless tobacco products. CSTHEA
further requires that individuals who manufacture, package, or import
smokeless tobacco products report to HHS the list of ingredients added
to tobacco in the manufacture of such products, and this requirement
has been implemented by a previous notice (59 FR 4714,).
In 1989 the smokeless tobacco industry submitted a business review
letter to the Department of Justice (DOJ), in accordance with 28 CFR
50.6. This letter requested approval of a collaborative industry effort
to develop a uniform analytical protocol for determining the nicotine
and moisture content of smokeless tobacco products.
In January 1993, DOJ extended permission to the smokeless tobacco
industry to develop a uniform analytical protocol for this purpose. A
work group representing the 10 major domestic manufacturers of
smokeless tobacco was convened on July 7, 1993. The workgroup developed
and submitted to CDC for approval the ``Protocol for Analysis of
Nicotine in Smokeless Tobacco Products.'' The protocol was revised by
CDC based on individual comments from peer reviewers and the National
Center for Environmental Health, CDC. The revised protocol, ``Protocol
for Analysis of Nicotine, Total Moisture, and pH in Smokeless Tobacco
Products,'' is hereafter referred to as the ``protocol.''
On May 2, 1997, a notice (62 FR 24115) was published in accordance
with the Paperwork Reduction Act to solicit public comment on the
proposed collection of data. A notice was also published (62 FR 24116,
May 2, 1997) to solicit public comment on the protocol. The protocol
consists of standard laboratory methods to measure nicotine, moisture,
and pH in smokeless tobacco products, and an equation (Henderson-
Hasselbalch) to calculate un-ionized nicotine. Nicotine is the major
alkaloid in tobacco and the drug in tobacco that causes addiction. In
the protocol, moisture is referred to as total moisture because the
method measures the amount of water and tobacco constituents in a
smokeless tobacco product that are volatile at temperatures of 99
degrees centigrade. pH is defined as the negative logarithm of the
molar concentration of hydrogen ions in an aqueous solution and is a
quantitative measure of acidity or alkalinity. The degree of nicotine
ionization is calculated from the Henderson-Hasselbalch equation. Un-
ionized nicotine is known to be the form of nicotine absorbed most
easily in the mouth. This protocol will provide CDC with information on
levels of nicotine found in smokeless tobacco products manufactured,
packaged, or imported during the previous calendar year. The schedule
for reporting this information to CDC corresponds to the reporting of
ingredients added to tobacco in the manufacture of smokeless tobacco
products November 8, 1994 (59 FR 55670,).
Following public request, on June 2, 1997, a notice (62 FR 29729)
was published extending the comment period on the proposed protocol by
an additional 30 days to July 2, 1997. A summary of the comments
received and CDC's response follows.
One respondent, on behalf of several smokeless tobacco
manufacturers, had several comments regarding the collection of data.
The respondent asserted that the protocols exceeded statutory
authorization by collecting pH and free base (hereinafter referred to
as ``un-ionized'', as reflected in the revised protocol) nicotine.
Furthermore, the respondent felt that the legislative history of CSTHEA
contemplates the reporting of nicotine content alone.
It is CDC's belief that the collection of pH of smokeless tobacco
products and the un-ionized nicotine content of each is authorized by
section 4 of CSTHEA. There is ample scientific evidence that mere
quantity of nicotine alone is insufficient in determining its effect on
a user; knowledge of pH and un-ionized nicotine content of the overall
nicotine quantity is essential in determining the rate of nicotine
absorption. pH and un-ionized nicotine content are essential factors
affecting nicotine bioavailability. Furthermore, Congress has never
defined exactly what it meant by ``quantity of nicotine'' in section
4(b) of CSTHEA. In light of ample scientific evidence indicating the
importance of pH and un-ionized nicotine content in assessing the
overall quantity of nicotine, CDC's interpretation of its statutory
authority is clearly permissible.
It is CDC's belief that the legislative history provides support
for requiring the reporting of moisture, pH, and un-ionized nicotine
content. The Senate

[[Page 14087]]

Report accompanying and establishing Congress' views on CSTHEA
repeatedly emphasized that ``it is essential that we inform the public
of the health effects of smokeless tobacco use and continue research on
such health effects as expeditiously as possible'' (Senate Report 99-
209, Dec. 4, 1985, p.13). Since scientific evidence has established
that knowledge of pH and un-ionized nicotine content is essential in
determining the health effects of smokeless tobacco, the reporting of
these elements is supported by the legislative history.
The respondent also commented that CDC failed to comply with
procedural obligations in violation of 5 USC Sec. 551 et seq., the
Administrative Procedure Act (APA). Specifically, the respondent
claimed that CDC failed to provide opportunity for advance review and
comment and failed to inform the public of the true nature of the
proposed protocol.
CDC feels that the notice given to the tobacco manufacturers was
clearly adequate under the APA. CDC's purpose in publishing the
protocol in the Federal Register was to solicit comments from the
public. This is the appropriate time for the manufacturers to review
the protocol and relay their comments to CDC, not before publication.
As a matter of courtesy, CDC has provided the manufacturers with a copy
of draft protocols before publication. CDC did that here as well, for
the tobacco companies were given an advance copy of the protocol before
formal publication. Therefore, CDC did provide the tobacco companies
with advance knowledge of the protocol, even though such notice was not
required.
Furthermore, CDC has not failed to inform the public of the true
nature of its proposed protocol. CDC sufficiently apprised the public
of the agency's legal authority to issue the proposed protocol, for it
explicitly states that it is operating under the authority of CSTHEA.
Thus, the public is on sufficient notice of the legal authority under
which CDC issued the proposed protocol, and has had full opportunity to
comment.
CDC is also not required to lay out potential criticisms of its
scientific positions in its notice and request for public comment. The
purpose of the notice and comment period is to provide interested
parties with the opportunity to conduct their own analysis of the
merits of the protocol, and to provide scientific or other criticisms,
if desired. CDC has clearly stated the terms and substance of its
proposed protocol as to provide the public with sufficient opportunity
to comment.
This respondent also commented that CDC failed to meet the
requirements of the Paperwork Reduction Act by failing to provide the
public with an accurate estimate of the burden of compliance. CDC
disagrees. CDC based its original estimate on the figures that were
submitted by the manufacturers themselves. Moreover, the 60-day notice
and comment period is designed to solicit comment on the accuracy of
the agency's estimate of the burden of the proposed collection of
information. 44 U.S.C. 3506(c)(2)(A)(ii). The tobacco manufacturers
felt that the estimate of burden had changed and commented as such at
that time. CDC then sought revised estimates from the manufacturers.
Only one manufacturer responded to the request for this information.
Based on this response, CDC conducted new analyses and revised its
estimate of burden accordingly. Thus, CDC clearly complied with the
Paperwork Reduction Act's requirements.
CDC also received comments regarding the protocol, which raised
technical and scientific concerns regarding collection and calculation
of the requested information. Several respondents supported the
protocol requirement of reporting not only moisture and nicotine
content but also pH and unionized nicotine levels. Support for this
aspect of the protocol was based on scientific evidence that the
nicotine-specific effects of a given amount of smokeless tobacco depend
as much on the pH of the product as on the nicotine content itself. CDC
agrees with this scientific observation regarding the utility of
determining smokeless tobacco pH and calculating unionized nicotine
levels in smokeless tobacco products.
One respondent, on behalf of several smokeless tobacco
manufacturers, stated that the protocols for nicotine analysis and
total moisture determination are scientifically flawed. Specifically,
the respondent stated that the Standards Addition Assay is flawed and
unnecessary, that the protocol specifies an unavailable vegetable-based
matrix, that triplicate determinations are unnecessary, and that the
protocol requires smokeless tobacco manufacturers to use a protocol
specific to cigarettes.
With respect to the Standards Addition Assay, CDC reaffirms the
function of the Standards Addition Assay and disputes the inadequacies
of the extraction testing offered by the respondent as a rationale for
eliminating the Standards Addition Assay. CDC revised the protocol to
facilitate preparation of a standard curve for the Standards Addition
Assay that encompasses the range of values expected from adding known
concentrations of nicotine to the smokeless tobacco product. Also, CDC
revised the protocol to specify when the Standards Addition Assay is to
be conducted by the testing facility.
Regarding use of a vegetable-based matrix, CDC acknowledges that a
nicotine-free tobacco surrogate is not readily available to serve as a
vegetable-based matrix; that is why the protocol thus specifies adding
known concentrations of nicotine to the smokeless tobacco product when
performing the Standards Addition Assay. CDC revised the text in
Endnote 1 of the protocol to eliminate the phrase ``routine testing of
random blind samples.''
Regarding triplicate determinations, CDC asserts that the potential
sources of smokeless tobacco product variability necessitate triplicate
determinations for evaluation of precision. In response to the comment
that CDC was attempting to ``bind the manufacturers'' to a cigarette
testing protocol in the smokeless tobacco testing protocol, CDC
clarifies that the disputed protocol is a sampling protocol, not a
testing protocol. Therefore, testing facilities should make use of the
document as reference material. However, for clarification, Endnote 11
of the protocol (Endnote 10 of the public comment version of the
protocol) was revised to read--``The testing facility must ensure that
samples are obtained through the use of a survey design protocol for
sampling `at one point in time' at the factory or warehouse. The survey
design protocol must address short-, medium-, and long-term smokeless
tobacco product variability (e.g., variability over time and from
container to container of the tobacco product) in a manner equivalent
to that described for cigarette sampling in Annex C of ISO Protocol
8243.''
This respondent also commented that the protocol for pH measurement
is scientifically flawed. Specifically, the respondent states that the
procedure is based on a nonvalidated protocol, that an inappropriate
volume of liquid is specified, that proper calibration of instruments
has not been incorporated in the protocol, that temperature is not
considered in the protocol, and that multiple pH measurements are
unnecessary.
CDC disagrees that the protocol for pH measurement is
scientifically flawed. The protocol to determine smokeless tobacco pH
is based on the validated protocol published by Henningfield et

[[Page 14088]]

al. (1995), which also provides the rationale for the quantity of
smokeless tobacco and the volume of liquid. Parameters that can be
standardized (sample size, sample preparation, quantity and purity of
standard and reagents, instrumentation, measurement time and
conditions, etc.) are specified in the protocol. Of note, the
Henningfield et al. (1995) reference was provided in the version of the
protocol that the respondent received for comment.
CDC agrees that careful calibration across the range of unknown
values to be measured is needed. The protocol was revised to read--
``Measure pH of sample after a two-point calibration of the pH meter to
an accuracy of two decimal places using standard pH buffers (4.01 and
7.00 or 7.00 and 10.00) that will encompass the expected pH value of
the smokeless tobacco product.''
CDC also agrees that conditions such as sample preparation, sample
size, extraction time, volume and purity of the water used, and
temperature must be controlled during determination of the pH of a
smokeless tobacco product. The protocol was revised to specify room
temperature for nicotine extraction and pH determination.
As described above, CDC recognizes that there are several potential
sources of smokeless tobacco product variability that necessitate
triplicate determinations for evaluation of precision. With respect to
pH determination, CDC recognizes the need for multiple measurements to
determine if pH values for the smokeless tobacco product vary
systematically with time. For edification, the protocol was revised to
read--``The first time pH values are determined for each lot of a
smokeless tobacco product, measure the pH of the smokeless tobacco
product at 5, 15, and 30 minutes. If there is no systematic variation
in pH values with time, all subsequent pH determinations for the lot
are made at 5 minutes. If there is systematic variation in pH values,
continue to measure the pH of the smokeless tobacco product until the
pH value is stable and does not vary more than 10% over 15 minutes.
Report the final pH value.''
This respondent further asserted that the ``smokeless tobacco pH''
theory has been discredited. In summary, the respondent states that
calculation of un-ionized nicotine is based on a discredited scientific
theory and that the ``smokeless tobacco pH'' theory ignores the
chemical, biological, and behavioral factors that govern absorption of
smokeless tobacco.
Un-ionized nicotine is known to be the form of nicotine most easily
absorbed in the mouth. pH determination is a component of the protocol
to allow calculation of un-ionized nicotine. The degree of nicotine
ionization is calculated from the Henderson-Hasselbalch equation. In a
document written at the request of the United States Tobacco Company,
Dr. Jeffrey R. Idle recognizes pH as one of the ``chemical factors
which determine the absorption of a substance dissolved in water across
a biological barrier with which the solution is in intimate contact.''
In the same document Dr. Idle concludes that ``The concept of a pH for
snuff depends upon a standardized and validated pH assay for aqueous
tobacco suspensions' and that ``The concept of pH for a moist solid
such as tobacco can only apply to a solution derived from a stirred
suspension of a standardized amount of tobacco in a standardized volume
of water.''
It is the intent of the protocol to provide smokeless tobacco
manufacturers with a ``standardized measurement'' of pH. Parameters
that can be standardized for pH, moisture, and nicotine determination
(sample size, sample preparation, quantity and purity of standards and
reagents, instrumentation, measurement time and conditions, etc.)--not
random conditions or circumstances unique to each smokeless tobacco
user such as ``residues of beverages'' in the mouth of the smokeless
tobacco user (chemical factors), ``surface area of the absorptive
tissues'' (biological factors), and ``expectoration'' or ``swallowing''
(behavioral factors)--are specified in the protocol. In addition, the
protocol's methodology is supported by the conclusions presented in a
recent review article that ``pH is a major determinant of nicotine
absorption across mucosal tissues'' in the mouth and that other
``behavioral and biological'' factors probably have ``little effect on
the rate of nicotine absorption'' (Tomar and Henningfield, Tobacco
Control 1997, 6:219-225).

Information Collection Provisions

This notice contains information collections which have been
approved by the Office of Management and Budget (OMB) under the
Paperwork Reduction Act of 1980 and assigned OMB Action Notice number
0920-0444 (expiration 01/31/2002). The title description, and
respondent information are shown below with an estimate of the
annualized costs and burden hours.
Title: Quantity of Nicotine Contained in Smokeless Tobacco Products
Manufactured, Imported, or Packaged in the United States.
Description: The Comprehensive Smokeless Tobacco Health Education
Act (CSTHEA) of 1986 requires HHS to collect this information. HHS is
authorized under CSTHEA to conduct research on the addictive nature of
nicotine and general health effects of using smokeless tobacco.
Description of Respondents: Businesses or other For-Profit
Organizations.
Estimates of Annualized Burden Hours and Costs: The average
annualized total cost to industry is $23,419. This is based on an
annualized estimated cost for 11 companies at $2,129 per company. Some
companies may choose to contract with an independent laboratory while
others may elect to complete the determinations in-house. For those
companies choosing to conduct the testing in-house, approximately
US$60,000 would be required to purchase the necessary equipment,
assuming none of the equipment was previously owned.
The annual response burden to the industry is estimated at 170
hours per smokeless tobacco company. Thus, for the 11 respondents, the
hour-burden is 1,870 hours.

--------------------------------------------------------------------------------------------------------------------------------------------------------
Avg. number of
Respondents Number of responses per Avg. hours per Estimated total Avg. cost per Estimated total
respond-ents respondent respondent hours respondent cost
--------------------------------------------------------------------------------------------------------------------------------------------------------
Tob. Mfrs......................................... 11 1 170 1,870 $2,129 $23,419
--------------------------------------------------------------------------------------------------------------------------------------------------------

Procedures to maintain confidentiality of nicotine, pH, and
moisture data: As provided by CSTHEA, HHS is required to treat the
nicotine, pH, and moisture information as a trade secret or
confidential in accordance

[[Page 14089]]

with 5 U.S.C. 552(b)(4)and 18 U.S.C. 1905. CSTHEA also requires HHS to
establish written procedures to assure the confidentiality of the
information provided. Consistent with these statutory provisions, HHS
has developed strict procedures for treating and protecting relevant
documents, including secured file storage and strictly-limited access
to the information. The procedures that are applicable to the nicotine
content of smokeless tobacco products comport with those already in
place for protecting the confidential lists of ingredients in
cigarettes and smokeless tobacco products. These procedures have proven
workable, effective, and acceptable to the companies required to report
the confidential information. The procedures, Guidelines for
Maintaining and Releasing Privileged Information Obtained in Accordance
With Sec. 4(b)(2)(a) of Public Law 99-252 (15 U.S.C. 4403), were
previously published in the February 1, 1994, Federal Register (59 FR
4714), and are available from CDC's Office on Smoking and Health upon
request.

Dated: March 17, 1999.
Martha Katz,
Acting Director, Centers for Disease Control and Prevention.

Protocol for Analysis of Nicotine, Total Moisture, and pH in
Smokeless Tobacco Products

I. Requirements ^{1, 2}

A. Reagents ³

1. Sodium hydroxide (NaOH), 2N
2. Methyl t-butyl ether (MTBE)
3. (-) -Nicotine (Fluka 72290) >99% purity ^{4, 5}
4. Quinoline (Aldrich)
5. Standard pH buffers; 4.01, 7.00, and 10.00
6. Deionized distilled water

B. Glassware and supplies

1. Volumetric flasks, class A
2. Culture tubes, 25 mm x 200 mm, with Teflon-lined screw caps
3. Pasteur pipettes
4. Repipettors (10 mL and 50 mL)
5. Linear shaker (configured to hold tubes in horizontal position)
^{6, 7}
6. Weighing dishes, aluminum
7. Teflon-coated magnetic stirring bars
8. Polypropylene containers, 50 mL

C. Instrumentation

1. Robot Coupe Model RSI 2V Scientific Batch Processor.
2. Capillary gas chromatograph, Hewlett Packard, Model 6890, with
split/splitless injector capability, flame ionization detector, and a
capillary column (Hewlett Packard HP-5, Crosslinked 5% PH ME Siloxane,
30 m length x 0.32 mm ID, film thickness 0.25 or 0.52 m).
3. Orion Model EA 940 pH meter equipped with Orion 8103 Ross
combination pH electrode.

D. Additional Equipment

Forced-air oven, Fisher Isotemp, regulated to 99
1.0 deg.C. Suggested dimensions: 18 x 18 x 20.''

E. Chromatographic Conditions ^{8, 9}

1. Detector temperature: 250 deg.C
2. Injector temperature: 250 deg.C
3. Flow rate at 100 deg.C--1.7 mL/min; with split ratio of 40:1
¹⁰
4. Injection volume: 2 l
5. Column conditions: 110-185 deg.C at 10 deg.C min ^-1; 185-
240 deg.C at 6 deg.C min ^-1, hold at final temperature for
10 min.

F. Sample Preparation ¹¹

There exist six different categories of commercial smokeless
tobacco products:

1. Dry snuff;
2. Moist (wet) snuff;
3. Moist (wet) snuff portion packs;
4. Plug;
5. Twist; and
6. Loose leaf.

Because of their physical characteristics, samples of three of the
six product categories must be ground before nicotine, total moisture,
and pH analyses can be conducted. The objective of grinding the samples
is to obtain a homogeneous sample with particles measuring
approximately 4 mm. Grinding to achieve this particle size should take
no more than 3 minutes. To ensure proper grinding and an adequate
amount of the ground sample for analysis, the minimum sample size of
all commercial products to be ground should not be less than 100 grams.
To ensure precision of analyses for nicotine, total moisture, and
pH, the samples that require grinding should be ground using a Robot
Coupe Model RSI 2V Scientific Batch Processor or its equivalent. This
is a variable speed (0 to 3000 RPM) processor. The variable speed motor
is required to ensure proper grinding of the tobacco tissues (and in
the case of pH determination, the moist (wet) snuff portion pack).
Elevated temperatures can result in moisture loss and an underestimated
value for moisture content. Hence, care must be taken during grinding
to avoid elevated temperatures. The bowl should be cleaned after each
grinding to obtain accurate results.
1. Dry snuff. Dry snuff samples do not need to be ground since the
product is a powder. The sample must be thoroughly mixed before
weighing for nicotine, total moisture, and pH analysis.
2. Moist (wet) snuff. Moist (wet) snuff samples do not need to be
ground. The sample must be thoroughly mixed before weighing for
nicotine, total moisture, and pH analysis.
3. Moist (wet) snuff portion packs. The tobacco contents of the
moist (wet) snuff portion packs do not need to be ground for nicotine,
total moisture, or pH analysis. The tobacco packaging material (the
``pouch'') should be separated from the tobacco and ground to obtain
particles measuring approximately 4 mm for pH analysis. The tobacco of
the moist (wet) snuff portion pack and the ground pouch are combined
and thoroughly mixed before pH analysis.
4. Plug tobacco. Break or cut apart plugs and add in portions to
grinder at 2000 RPM. Reduce RPM or stop grinding if sample bowl becomes
warm. Pulse the Robot Coupe, when needed, to complete grinding. Grind
samples until approximately 4 mm in size. The total grinding time
should be no more than 3 minutes.
5. Twist tobacco. Separate twists, add to grinder and grind at 2000
RPM. Reduce RPM or stop grinding if sample bowl becomes warm. Continue
grinding until sample particles are approximately 4 mm in size. The
total time for grinding should be no more than 3 minutes.
6. Loose leaf. Grind in the same manner as described in 4 and 5 to
obtain product with particle size of approximately 4 mm.

II. Nicotine Analysis ¹²

A. Calibration Standards

1. Internal Standard (IS)
Weigh 10.00 grams of quinoline, transfer to a 250 mL volumetric
flask and dilute to volume with MTBE. This solution will be used for
calibration of the instrument for the nicotine calibration curve
(II.A.2), for the standards addition assay (II.B), and for preparation
of the extracting solution (II.D).
2. Nicotine Calibration Curve
a. Weigh 1.0000 gram of nicotine into a clean, dry 100 mL
volumetric flask and dilute to volume with MTBE. This gives a nicotine
concentration of 10 mg/mL for the stock solution.
b. Accurately pipette 0.5 mL of IS from stock solution (II.A.1) to
five clean, dry 50 mL volumetric flasks. To prepare a nicotine standard
corresponding to a concentration of 0.8 mg/mL, pipette exactly 4.0 mL
of the nicotine standard

[[Page 14090]]

(II.A.2.a) to a 50 mL volumetric flask containing the internal standard
and dilute to volume with MTBE. To obtain nicotine concentrations
equivalent to 0.6, 0.4, 0.2, and 0.1 mg/mL, pipette precisely 3.0, 2.0,
1.0, and 0.5 mL, respectively, of the nicotine standard into the four
remaining flasks and dilute to volume with MTBE.
c. Transfer aliquots of the five standards to auto sampler vials
and determine the detector response for each standard using gas
chromatographic conditions described in I.E.
d. Calculate least squares line for linear equation from these
standards by obtaining the ratio of Areanicotine/
AreaIS. This ratio will be the Y value and the concentration
of nicotine will be the X value for determining the linear equation of
the line (Equation 1):

Equation 1:
Y = a + bX;

Where:

X = Concentration of nicotine in mg
Y =Areanicotine/AreaIS
a = intercept on the ordinate (y axis)
b = slope of the curve

The final result will be reported in the following units:
Concentration of nicotine = mg of nicotine/gram of tobacco sample.
e. Determine the recovery of nicotine by pipetting 10 mL of the 0.4
mg/mL nicotine standard to a screw capped tube containing 1.0 mL of 2 N
NaOH. Cap the tube. Shake the contents vigorously and allow the phases
to separate. Transfer an aliquot of the organic phase to an injection
vial and inject. Calculate the concentration of nicotine using the
equation of the line in II.A.2.d above. This should be repeated two
more times to obtain an average of the three values. The recovery of
nicotine can be obtained by using the following equation:

Equation 2:
Recovery = Nicotinecalculated/Nicotineactual

B. Standards Addition Assay

Prior to analyzing a smokeless tobacco product for nicotine
content, the testing facility must validate the system to verify that
matrix bias is not occurring during nicotine extraction. This is done
by analyzing the nicotine calibration standards in the same vegetable
matrix as the smokeless tobacco. The first time each lot of a smokeless
tobacco product is evaluated, the Standards Addition Assay will be
performed, and documentation of its performance and of the nicotine
concentrations selected for the standard curve (II.B.2) will be
submitted to the Centers for Disease Control and Prevention.
1. Using an analytical balance, accurately weigh 1.000
0.020 gram of the homogeneous, prepared tobacco sample into a culture
tube. Repeat this five times for a total of 6 culture tubes containing
the smokeless tobacco product. Record the weight of each sample.
2. Prepare a five-point standard curve for the Standards Addition
Assay. The standard curve must consist of nicotine concentrations that
encompass the range of values expected from adding known concentrations
of the nicotine standard (II.A.2.a) to a measured quantity of the
smokeless tobacco product (1.000 0.020 gram, described in
II.B.1.). The sixth culture tube is not supplemented with nicotine and
serves as an analytical blank. Allow the samples to equilibrate for 10
minutes.
3. Pipette 5 mL of 2 N NaOH into each tube. Cap each tube. Swirl to
wet sample and allow to stand 15 minutes.\13\
4. Pipette 50 mL of extraction solution (II.D.1) into each tube.
Cap each tube and tighten.\14\
5. Place tubes in rack(s), place racks in linear shaker in
horizontal position and shake for two hours.
6. Remove rack(s) from shaker and place in vertical position to
allow the phases to separate.
7. Allow the solvent and nicotine supplemented samples and the
blank to separate (maximum 2 hours).
8. Transfer aliquots of the five standards and the blank from the
extraction tubes to sample vials and determine the detector response
for each using gas chromatographic conditions described in I.E.
9. Subtract the Areanicotine/AreaIS of the
blank from the Areanicotine/AreaIS of each of the
standards.
10. Calculate least squares line for linear equation from the
corrected standards as described above (Equation 1) in II.A.2.d.
The final corrected result will be reported in the following units:
Concentration of nicotine = mg of nicotine/gram of tobacco sample.
11. Determine the recovery of nicotine by pipetting 10 mL of the
0.4 mg/mL nicotine standard to a screw capped tube containing 1.0 mL of
2 N NaOH and 10 mL of extraction solution (II.D.1). Cap the tube and
tighten. Shake the contents vigorously and allow the phases to
separate. Transfer an aliquot of the organic phase to an injection vial
and inject. Calculate the concentration of nicotine using the equation
of the line above in II.A.2.d. This should be repeated two more times
to obtain an average of the three values. The recovery of nicotine can
be obtained by using Equation 2:

Recovery = Nicotinecalculated/Nicotineactual

12. Compare the results of steps II.A.2. and II.B. If they differ
by a factor of 10% or more, the recovery of nicotine from the aqueous
matrix is not equivalent to recovery from the vegetable matrix of the
smokeless tobacco product. In this instance, the nicotine concentration
of the smokeless tobacco product must be determined from a nicotine
calibration curve prepared from nicotine standards in a vegetable-based
matrix.

C. Quality Control Pools

At least two quality control pools at the high and low ends of the
expected nicotine values are recommended to be included in each
analytical run. The pools should be analyzed in duplicate in every run.
The quality control pools should be available in sufficient quantity to
last for all analyses of a product lot.

D. Sample Extraction Procedure \12\

1. Extraction solution is prepared by pipetting 10 mL of the IS
from the stock solution (II.A.1) to a 1000 mL volumetric flask and
diluting to volume with MTBE.
2. Using an analytical balance, accurately weigh 1.000
0.020 gram of prepared tobacco sample into culture tube and record
weight.\15\ The number of products sampled per lot should reflect an
acceptable level of precision.\16\ The test material is to be
representative of the product that is sold to the public and therefore
should consist of sealed, packaged samples from each lot of finished
product that is ready for commercial distribution. Triplicate
determinations will provide precision data.
3. Pipette 5 mL of 2 N NaOH into the tube. Cap the tube. Swirl to
wet sample and allow to stand 15 minutes.\13\
4. Pipette 50 mL of extraction solution into tube, cap tube and
tighten.\14\
5. Place tubes in rack(s), place racks in linear shaker in
horizontal position and shake for two hours.
6. Remove rack(s) from shaker and place in vertical position to
allow the phases to separate.
7. Allow the solvent and sample to separate (maximum 2 hours).
Transfer an aliquot from the extraction tube to a sample vial and cap.
8. Analyze the extract using GC conditions as described above (I.E)
and calculate the concentration of nicotine using the linear
calibration equation. Correct percent nicotine values for both recovery
and weight of sample by using Equation 3.\17\

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9. Report the final nicotine determination as mg of nicotine per
gram of the tobacco product (mg nicotine/gram), to an accuracy level of
two decimal places for each lot and for each brand name (e.g., Skoal
Bandits Wintergreen, Skoal Long Cut Cherry, Skoal Long Cut Wintergreen,
etc.). All data should include the mean value with a 95% confidence
interval, the range of values, the number of samples tested per lot,
the number of lots per brand name, and the estimated precision of the
mean. Information will be reported for each manufacturer and variety
(including brand families and brand variations) and brand name (e.g.,
Skoal Bandits Wintergreen, Skoal Long Cut Cherry, Skoal Long Cut
Wintergreen, etc.).

III. Total Moisture Determination

A. This procedure is a modification of AOAC Method 966.02 (1990)
and is referred to as ``Total Moisture Determination'' because it
determines water and tobacco constituents that are volatile at
temperatures of 99 1.0 deg.C.
B. Accurately weigh 5.00 grams of the sample (ground to pass
4 mm screen) \19\ into a weighed moisture dish and place
uncovered dish in oven.\20\ The number of products sampled per lot
should reflect an acceptable level of precision.\16\ The test material
is to be representative of the product that is sold to the public and
therefore should consist of sealed, packaged samples from each lot of
finished product that is ready for commercial distribution. Triplicate
determinations will provide precision data.
C. Do not exceed 1 sample/10 sq in. (650 sq cm) shelf space, and
use only 1 shelf. Dry 3 hr at 99 1.0 deg.C. Remove from
oven, cover, and cool in desiccator to room temp. (about 30 min).
Reweigh and calculate percent moisture.
D. Report the final moisture determination as a percentage (%), to
an accuracy level of one decimal place for each lot and for each brand
name (e.g., Skoal Bandits Wintergreen, Skoal Long Cut Cherry, Skoal
Long Cut Wintergreen, etc.). All data should include the mean value
with a 95% confidence interval, the range of values, the number of
samples tested per lot, the number of lots per brand name, and the
estimated precision of the mean. Information will be reported for each
manufacturer and variety (including brand families and brand
variations) and brand name (e.g., Skoal Bandits Wintergreen, Skoal Long
Cut Cherry, Skoal Long Cut Wintergreen, etc.).

IV. pH Measurement ^12,21

A. Test samples as soon as possible after they are received. The
number of products sampled per lot should reflect an acceptable level
of precision.\16\ The test material is to be representative of the
product that is sold to the public and therefore should consist of
sealed, packaged samples from each lot of finished product that is
ready for commercial distribution. Triplicate determinations will
provide precision data.
B. Accurately weigh 2.00 grams of the sample. Place in a 50 mL
polypropylene container with 10 mL deionized distilled water.
C. Place Teflon-coated magnetic stirring bar in container and stir
mixture continuously throughout testing.
D. Measure pH of sample after a two-point calibration of the pH
meter to an accuracy of two decimal places using standard pH buffers
(4.01 and 7.00 or 7.00 and 10.00) that will encompass the expected pH
value of the smokeless tobacco product.
E. The first time pH values are determined for each lot of a
smokeless tobacco product, measure the pH of the smokeless tobacco
product at 5, 15, and 30 minutes. If there is no systematic variation
in pH values with time, all subsequent pH determinations for the lot
are made at 5 minutes. If there is systematic variation in pH values,
continue to measure the pH of the smokeless tobacco product until the
pH value is stable and does not vary more than 10% over 15 minutes.
Report the final pH value.
F. Report the final pH determination to an accuracy level of two
decimal places for each lot and for each brand name (e.g., Skoal
Bandits Wintergreen, Skoal Long Cut Cherry, Skoal Long Cut Wintergreen,
etc.). All data should include the mean value with a 95% confidence
interval, the range of values, the number of samples tested per lot,
the number of lots per brand name, and the estimated precision of the
mean. Information will be reported for each manufacturer and variety
(including brand families and brand variations) and brand name (e.g.,
Skoal Bandits Wintergreen, Skoal Long Cut Cherry, Skoal Long Cut
Wintergreen, etc.).
G. Estimate the un-ionized (free) nicotine content with the
Henderson-Hasselbalch equation (Equation 4), based on measured pH and
nicotine content.

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H. Report the final estimated un-ionized (free) nicotine as a
percentage (%) of the total nicotine content, to an accuracy level of
two decimal places and as mg of un-ionized (free) nicotine per gram of
the tobacco product (mg un-ionized (free) nicotine/gram), to an
accuracy level of two decimal places for each lot and for each brand
name (e.g., Skoal Bandits Wintergreen, Skoal Long Cut Cherry, Skoal
Long Cut Wintergreen, etc.). All data should include the mean value
with a 95% confidence interval, the range of values, the number of
samples tested per lot, the number of lots per brand name, and the
estimated precision of the mean. Information will be reported for each
manufacturer and variety (including brand families and brand
variations) and brand name (e.g., Skoal Bandits Wintergreen, Skoal Long
Cut Cherry, Skoal Long Cut Wintergreen, etc.).

Billing Code 4163-18-P
Sample calculation:

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V. Assay Criteria for Quality Assurance

A. Establishing Limits for Quality Control Parameters

All quality control parameters must be determined within the
laboratory in which they are to be used. At least 10 within-laboratory
runs must be performed to establish temporary confidence intervals for
the quality control parameters. Permanent limits should be established
after 20 runs and should be reestablished after each additional 20
runs.

B. Exclusion of Outliers From the Calibration Curve \18\

The coefficient of determination between Areanicotine/
AreaIS and nicotine concentration should be equal to 0.99 or
higher. Any calibration standard having an estimated concentration
computed from the regression equation (Equation 1) which is different
from its actual concentration by a factor of 10% can be excluded from
the calibration curve. Up to two concentrations may be excluded, but
caution should be used in eliminating values, since bias may be
increased in the calibration curve. If an outlier value is eliminated,
its duplicate value must also be discarded to avoid producing a new
bias. All unknowns must fall within the calibration curve; therefore,
duplicate values excluded at either end of the calibration curve will
restrict the useful range of the assay.

C. Quality Control Pools and Run Rejection Rules

The mean estimated nicotine concentration in a pool should be
compared with the established limits for that pool based on at least 20
consecutive runs. An analytical run should be accepted or rejected
based upon the following set of rules adapted from Westgard et al.
(1981).
1. When the mean of one QC pool exceeds the limit of x
3 standard deviations (SD), then the run is rejected as out of control.
Here, x and SD represent the overall mean and standard deviation of all
estimated nicotine concentrations for a particular pool in the runs
which were used to establish the control limits.
2. When the mean nicotine concentrations in two QC pools in the
same run exceed the same direction, then the run must be rejected. The
same direction is the condition in which both pools exceed either the x
+ 2 SD or the x -2 SD limits.
3. When the mean nicotine concentrations in one or two QC pools
exceed their x 2 SD limits in the same direction in two
consecutive runs, then both runs must be rejected.
4. When the mean nicotine concentrations in two QC pools are
different by more than a total of 4 SD, then the run must be rejected.
This condition may occur, for example, when one QC pool is 2 SD greater
than the mean, and another is 2 SD less than the mean.

Endnotes

The comments and notes listed below can be described as Good
Laboratory Practice guidelines; they are described in detail in this
protocol to ensure minimal interlaboratory variability in the
determination of nicotine, total moisture, and pH in smokeless
tobacco.
\1\ This protocol assumes that the testing facility will
implement and maintain a stringent Quality Assurance/Quality Control
program to include, but not be limited to, regular interlaboratory
comparisons, determination of the quality and purity of purchased
products, and proper storage and handling of all reagents and
samples.
\2\ When a specific product or instrument is listed, it is the
product or instrument that was used in the development of this
method. Equivalent products or instruments may also be used. Use of
trade names is for identification only and does not constitute
endorsement by the Public Health Service or the U.S. Department of
Health and Human Services.
\3\ All chemicals, solvents, and gases are to be of the highest
purity.
\4\ Companies must ensure that the purity of the nicotine base
is certified by the vendor and that the chemical is properly stored.
However, nicotine base oxidizes with storage, as reflected by the
liquid turning brown. If oxidation has occurred, the nicotine base
should be distilled prior to use in making a standard solution.
\5\ A suggested method for the determination of nicotine purity
is CORESTA Recommended Method No. 39.
\6\ Horizontal shaking will allow more intimate contact of this
three phase extraction. There is a minimal dead volume in the tube
due to the large sample size and extraction volume. This
necessitates horizontal shaking.
\7\ If linear shaker is not available, a wrist action shaker
using 250 mL stoppered Erlenmeyer flasks can be substituted. Values
for nicotine are equivalent to those obtained from the linear
shaker.
\8\ After installing a new column, condition the column by
injecting a tobacco sample extract on the column, using the
described column conditions. Injections should be repeated until
areas of IS and nicotine are reproducible. This will require
approximately four injections. Recondition column when instrument
has been used infrequently and after replacing glass liner.
\9\ Glass liner and septum should be replaced after every 100
injections.
\10\ Most older instruments operate at constant pressure. To
reduce confusion, it is suggested that the carrier gas flow through
the column be measured at the initial column temperature.
\11\ The testing facility must ensure that samples are obtained
through the use of a survey design protocol for sampling ``at one
point in time'' at the factory or warehouse. The survey design
protocol must address short-, medium-, and long-term smokeless
tobacco product variability (e.g., variability over time and from
container to container of the tobacco product) in a manner
equivalent to that described for cigarette sampling in Annex C of
ISO Protocol 8243. Information accompanying results for each sample
should include, but not be limited to:
1. For each product--manufacturer and variety (including brand
families and brand variations) and brand name (e.g., Skoal Bandits,
Skoal Long Cut Cherry, Skoal Long Cut Wintergreen, etc.)
information.
2. Product ``category,'' e.g., loose leaf, plug, twist, dry
snuff, moist (wet) snuff, etc.
3. Lot number.
4. Lot size.
5. Number of randomly sampled, sealed, packaged (so as to be
representative of the product that is sold to the public) smokeless
tobacco products selected per lot (sampling fraction) for nicotine,
moisture, and pH determination.
6. Documentation of method used for random sample selection.
7. ``Age'' of product when received by testing facility and
storage conditions prior to analysis.
\12\ Extraction of nicotine and pH determination must be
performed with reagents and samples at a room temperature of 22-
25 deg.C. Room temperature should not vary more than 1 deg.C during
extraction of nicotine or pH determination.
\13\ Use non-glass 10 mL repipette for transferring NaOH
solution.
\14\ Use 50 mL repipette for transferring MTBE.
\15\ For dry snuff, use 0.500 0.010 gram sample.
\16\ The testing facility is referred to ISO Procedure 8243 for
a discussion of sample size and the effect of variability on the
precision of the mean of the sample (ISO 8243, 1991).
\17\ When analyzing new smokeless tobacco products, extract
product without IS to determine if any components co-elute with the
IS or impurities in the IS. This interference could artificially
lower calculated values for nicotine.
\18\ The calculated nicotine values for all samples must fall
within the low and high nicotine values used for the calibration
curve. If not, prepare a fresh nicotine standard solution and an
appropriate series of standard nicotine dilutions. Determine the
detector response for each standard using chromatographic conditions
described in I.E.
\19\ The method is a modification of AOAC Method 966.02 (1990)
in that the ground tobacco passes through a 4 mm screen rather than
a 1 mm screen.
\20\ When drying samples, do not dry different products (e.g.,
moist (wet) snuff, dry snuff, loose leaf) in the oven at the same
time since this will produce errors in the moisture determinations.
\21\ The method is based on a method published by Henningfield
et al. (1995).

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References

AOAC (Association of Official Analytical Chemists). Official
Methods of Analysis. 966.02: Moisture in Tobacco. (1990) Fifth
Edition. K. Helrich (ed). Association of Official Analytical
Chemists, Inc. Suite 400, 2200 Wilson Boulevard, Arlington, Virginia
22201 USA.
CORESTA (Centre de Coopration pour les Recherches Scientifiques
relatives au Tabac). Recommended Method No. 39: Determination of the
purity of nicotine and nicotine salts by gravimetric analysis--
Tungstosilic acid method November, 1994. 87-90.
CRC Handbook of Chemistry and Physics. R.C. Weast, D.R. Lide,
M.J. Astle, and WH. Beyer (eds). 70th ed. Boca Raton, Florida: CRC
Press (1989-1990) D-162.
Henningfield, J.E., Radzius A., Cone E.J. (1995). Estimation of
available nicotine content of six smokeless tobacco products.
Tobacco Control 4:57-61.
ISO (International Organization for Standardization). IOS 8243:
Cigarettes--Sampling. (1991). Second Edition. Prepared by Technical
Committee ISO/TC 126, Tobacco and tobacco products. International
Organization for Standardization, Case Postale 56, CH-1211 Genve 20,
Switzerland.
Westgard J.O., Barry P., Hunt M., and Groth T. (1981). A multi-
rule Shewhart chart for quality control in clinical chemistry.
Clinical Chemistry 27:493.

[FR Doc. 99-7022 Filed 3-22-99; 8:45 am]
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